Genome-wide landscape of ApiAP2 transcription factors reveals a heterochromatin-associated regulatory network duringPlasmodium falciparumblood-stage development

Abstract

Heterochromatin-associated gene silencing controls multiple physiological processes in malaria parasites, however, little is known concerning the regulatory network and cis-acting sequences involved in the organization of heterochromatin and how they modulate heterochromatic gene expression. Based on systematic profiling of genome-wide occupancy of eighteen Apicomplexan AP2 transcription factors by ChIP-seq analysis, we identify and characterize eight heterochromatin-associated factors (PfAP2-HFs), which exhibit preferential enrichment within heterochromatic regions but with differential coverage profiles. Although these ApiAP2s target euchromatic gene loci via specific DNA motifs, they are likely integral components of heterochromatin independent of DNA motif recognition. Systematic knockout screenings of ApiAP2 factors coupled with RNA-seq transcriptomic profiling revealed three activators and three repressors of heterochromatic gene expression including four PfAP2-HFs. Notably, expression of virulence genes is either completely silenced or significantly reduced upon the depletion of PfAP2-HC. Integrated multi-omics analyses reveal autoregulation and feed-forward loops to be common features of the ApiAP2 regulatory network, in addition to the occurrence of dynamic interplay between local chromatin structure and ApiAP2s in transcriptional control. Collectively, this study provides a valuable resource describing the genome-wide landscape of the ApiAP2 family and insights into functional divergence and cooperation within this family during the blood-stage development of malaria parasites.

Identification of Antimalarial Compounds that Inhibit Apicomplexan AP2 Proteins

“…AP2-EXP occupancy is well correlated with the activating chromatin reader PfBDP1 15 . This coincides with results reported for PfAP2-I, which binds many of the same genomic loci as AP2-EXP and BDP1 11,29,32 . Together, these results point to transcription activation via chromatin modulation as a key role of AP2-EXP in the asexual blood stage.…”

“…Unexpectedly, AP2-EXP target genes identified in our study overlap poorly with the differentially regulated transcripts reported as a result of truncation of the AP2-EXP protein (Table S5) 33 . There is greater overlap between our study and the AP2-EXP ChIP-seq in trophozoites recently described by Shang et al 29 , with 50/101 target genes overlapping (Table S5) . It is therefore possible that the truncated AP2-EXP retains some critical function that relies on DNA binding.…”

“…Since AP2-EXP is predicted to act as a transcription factor 29,33 we hypothesized that changes in parasite transcript abundance should occur when AP2-EXP DNA binding is competed by Compound C. To test this hypothesis, we cultured parasites in 12µM (0.66xIC 50 ) Compound C or DMSO vehicle and measured comparative RNA abundance by DNA microarray at seven time points during the asexual blood stage. As a quality control for normal IDC progression, we found a high correlation between a set of “control” genes 52,53 with an established 48-hour periodicity with maxima at different times throughout the 3D7 P. falciparum IDC 6 in our DMSO control and Compound C mRNA abundance profiles (Fig S12A-D) .…”

“…A ‘no epitope control’ ChIP done on a wild type 3D7 parasite line with the anti-GFP antibody detected only 2 peaks, neither of which overlapped with those of AP2-EXP::GFP or AP2-EXP::HA. Both the conserved AP2-EXP peaks and individual replicates were highly enriched for the known AP2-EXP and PbAP2-Sp DNA sequence motif CATGCA 29,58 (Fig 5C, Fig S15A-C).…”

“…Their plant-like origin distinguishes them from all other known transcription factors in humans or mosquitoes 20 . Over half of the ApiAP2 proteins are predicted to be essential for the IDC in P. falciparum based on a random mutagenesis screen 26 , and in targeted knockout screens only 11/23 ApiAP2 coding sequence disruptions attempted were successful [27][28][29] . In the rodent malaria model species P. berghei 30 and P. yoelii 31 , 11 ApiAP2 proteins have developmental lethal phenotypes in the sexual blood or mosquito stages.…”